Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice - effects of peripheral axotomy or hindpaw inflammation.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

Neurotoxic (+)-methamphetamine treatment in rats increases brain-derived neurotrophic factor and tropomyosin receptor kinase B expression in multiple brain regions.

Methamphetamine (MA) is an abused stimulant which can result in cognitive deficits and monoamine depletions. Animal models of neurotoxic MA exposure show reductions in dopamine, serotonin, and their associated transporters. MA abuse can result in long-term attention, working memory, and executive function deficits in humans and deficits in route-based egocentric learning, novel object recognition, and novel odor preference in rodents. MA has also been shown to affect brain-derived neurotrophic factor (BDNF) in humans and rodents. This experiment examined the effects of a MA binge dosing regimen (10 mg/kg x 4 at 2 h intervals, s.c.) in Sprague-Dawley rats on BDNF, tropomyosin receptor kinase B (TrkB), and tyrosine hydroxylase (TH) mRNA expression, and plasma corticosterone. Tissues were collected 1, 7, and 24 h following the last MA dose. Expression of BDNF and TrkB mRNA was analyzed using in situ hybridization with cRNA probes. Frontal, parietal, and entorhinal cortical BDNF mRNA expression were increased by MA exposure at all time-points. Increases in BDNF mRNA were also seen in the hippocampal CA1, prefrontal cortex (PFC), piriform cortex, and locus coeruleus but only at specific times. TrkB mRNA expression was modified in several subregions of the hippocampus as well as in PFC and striatum. TH mRNA was increased at the 1 h time-point in the substantia nigra pars compacta with no differences noted at the other times. Corticosterone levels were increased at all three time-points. The findings suggest that BDNF and its receptor may be upregulated as a compensatory mechanism after MA exposure.

Decreased expression of ErbB4 and tyrosine hydroxylase mRNA and protein in the ventral midbrain of aged rats.

Decreased availability or efficacy of neurotrophic factors may underlie an increased susceptibility of mesencephalic dopaminergic cells to age-related degeneration. Neuregulins (NRGs) are pleotrophic growth factors for many cell types, including mesencephalic dopamine cells in culture and in vivo. The functional NRG receptor ErbB4 is expressed by virtually all midbrain dopamine neurons. To determine if levels of the NRG receptor are maintained during aging in the dopaminergic ventral mesencephalon, expression of ErbB4 mRNA and protein was examined in young (3 months), middle-aged (18 months), and old (24-25 months) Brown Norway/Fischer 344 F1 rats. ErbB4 mRNA levels in the substantia nigra pars compacta (SNpc), but not the adjacent ventral tegmental area (VTA) or subtantia nigra pars lateralis (SNl), were significantly reduced in the middle-aged and old animals when compared to young rats. Protein expression of ErbB4 in the ventral midbrain was significantly decreased in the old rats when compared to the young rats. Expression of tyrosine hydroxylase (TH) mRNA levels was significantly reduced in the old rats when compared to young animals in the SNpc, but not in the VTA or SNI. TH protein levels in the ventral midbrain were also decreased in the old animals when compared to the young animals. These data demonstrate a progressive decline of ErbB4 expression, coinciding with a loss of the dopamine-synthesizing enzyme TH, in the ventral midbrain of aged rats, particularly in the SNpc. These findings may implicate a role for diminished NRG/ErbB4 trophic support in dopamine-related neurodegenerative disorders of aging such as Parkinson's disease.

Environmental enrichment attenuates cognitive deficits, but does not alter neurotrophin gene expression in the hippocampus following lateral fluid percussion brain injury.

Environmental enrichment attenuates neurological deficits associated with experimental brain injury. The molecular events that mediate these environmentally induced improvements in function after injury are largely unknown, but neurotrophins have been hypothesized to be a neural substrate because of their role in cell survival and neural plasticity. Furthermore, exposure to complex environments in normal animals increases neurotrophin gene expression. However, following an ischemic injury, environmental enrichment decreases neurotrophin mRNA levels. Whether these contrasting findings are attributable to differences between injured and uninjured animals or are dependent upon the specific type of brain injury has not been determined. We examined the effects of 14 days of environmental enrichment following a lateral fluid percussion brain injury on behavior and gene expression of brain-derived neurotrophic factor, its high-affinity receptor, TrkB, and neurotrophin-3 in the rat hippocampus. Environmental enrichment attenuated learning deficits in the injured animals, but neither the injury nor housing conditions influenced neurotrophin/receptor mRNA levels. From these data we suggest that following brain trauma, improvements in learning associated with environmental enrichment are not mediated by alterations in brain-derived neurotrophic factor, TrkB or neurotrophin-3 gene expression.

Neuropeptides: brain messengers of many faces.

Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.

Effects of chronic ethanol administration on expression of BDNF and trkB mRNAs in rat hippocampus after experimental brain injury.

Previous evidence indicates that both chronic alcohol treatment and traumatic brain injury modulate expression of certain neurotrophins and neurotrophin receptors in cortical tissue. However, the combined effects of chronic alcohol and brain trauma on expression of neurotrophins and their receptors have not been investigated. In the present study, we examined the effects of 6 weeks of chronic ethanol administration on lateral fluid percussion (FP) brain injury-induced alterations in expression of mRNAs for the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor, trkB, in rat hippocampus. In both the control- (pair-fed isocaloric sucrose) diet and the chronic ethanol-diet groups, unilateral FP brain injury induced a bilateral increase in levels of both BDNF and trkB mRNAs in the dentate gyrus granule cell layer, and of BDNF mRNA in hippocampal region CA3. However, no significant differences in expression were found between the control-diet and ethanol-diet groups, in either the sham-injured or FP-injured animals. These findings suggest that 6 weeks of chronic ethanol administration does not alter the plasticity of hippocampal BDNF/trkB expression in response to experimental brain injury.

Differential expression of neurotrophin and neurotrophin receptor mRNAs in and adjacent to fetal midbrain grafts implanted into the dopamine-denervated striatum.

This study examined the expression of neurotrophins and neurotrophin receptors in the lesion/transplanted striatum at four different time points after transplantation. The ventral mesencephalic region was dissected from a single rat fetus at embryonic day 14 (E14) and implanted into the denervated striatum of rats with unilateral 6-hydroxydopamine lesions. Transplanted rats were killed at 1, 2, 3, or 4 weeks after transplantation surgery and the brains subsequently prepared for semiquantitative in situ hybridization analysis of neurotrophin and neurotrophin trk receptors. Hybridization of cRNA probes for trkB or trkC showed a time-dependent reduction within the transplant during the first 4 weeks after transplantation; hybridization of brain-derived neurotrophic factor or tyrosine hydroxylase mRNA probes within the transplant did not change significantly during the same posttransplantation period. Hybridization of the trkB mRNA probe in host striatum adjacent to the transplant was significantly higher than probe hybridization in the corresponding region of the intact striatum during the first 2 weeks after transplantation, but by the 3rd and 4th week, probe hybridization in the denervated/transplanted and intact striatum were the same. Lesioned animals without transplants maintained higher trkB mRNA probe hybridization in the denervated striatum than in the intact striatum at the same postlesion time points suggesting that lesioned/transplanted animals show a normalization of trkB mRNA probe hybridization. Hybridization of the trkC mRNA probe in the lesioned/transplanted striatum was significantly lower than that observed in the intact striatum 4 weeks after transplantation; however, at this same time point we observed a similar reduction of trkC probed hybridization in lesioned animals without transplants. The results of the study show dynamic neurotrophic activity occurring within the transplant and host tissue during the first month of transplant development.

Multiple messengers in descending serotonin neurons: localization and functional implications.

In the present review article we summarize mainly histochemical work dealing with descending bulbospinal serotonin neurons which also express a number of neuropeptides, in particular substance P and thyrotropin releasing hormone. Such neurons have been observed both in rat, cat and monkey, and may preferentially innervate the ventral horns of the spinal cord, whereas the serotonin projections to the dorsal horn seem to lack these coexisting peptides. More recent studies indicate that a small population of medullary raphe serotonin neurons, especially at rostral levels, also synthesize the inhibitory neurotransmitter gamma-amino butyric acid (GABA). Many serotonin neurons contain the glutamate synthesizing enzyme glutaminase and can be labelled with antibodies raised against glutamate, suggesting that one and the same neuron may release several signalling substances, causing a wide spectrum of post- (and pre-) synaptic actions.

Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells.

The epidermal growth factor receptor family consists of four related tyrosine kinases: the epidermal growth factor receptor (EGF-R or ErbB), ErbB2, ErbB3, and ErbB4. These receptors are capable of extensive cross-activation upon the binding of their ligands - the EGF family of peptides for EGF-R and the neuregulins for ErbB3 and ErbB4. Since EGF-R is expressed by proliferating cells in the central nervous system (CNS), including multipotent CNS stem cells, we examined the expression of ErbB2, ErbB3 and ErbB4 in the germinal epithelia of the developing rat brain using in situ hybridization. ErbB2 and ErbB4 mRNAs were widely distributed within the germinal zones as early as E12. However, as development proceeded, ErbB2 mRNA was mainly present within the layers of cells immediately adjacent to the ventricular surface - the ventricular zone, while ErbB4 mRNA was predominantly expressed by subventricular zone cells, in the regions where these specialized germinal epithelia were present. ErbB3 mRNA distribution within germinal epithelia was more restricted, primarily confined to the diencephalon and rostral midbrain. Cultured neurospheres, which contain CNS stem cells, expressed ErbB2, ErbB4 and, to a lesser extent, ErbB3 protein as demonstrated by Western blot analysis. This expression declined during following differentiation. Heregulin-beta1, a neuregulin, had no effect on the proliferative capacity of neurospheres. Overall, our results indicate that ErbB2, ErbB3 and ErbB4 may play important and distinct roles in the genesis of the CNS. However, our in vitro data do not support a role for neuregulins in proliferation, per se, of CNS stem cells.

Multiple trophic actions of heparin-binding epidermal growth factor (HB-EGF) in the central nervous system.

The epidermal growth factor (EGF) family of ligands interacts with the epidermal growth factor receptor (EGF-R) to produce numerous direct and indirect actions on central nervous system cells. They induce the proliferation of astrocytes and multipotent progenitors ('stem' cells) and promote the survival and differentiation of postmitotic neurons. Heparin-binding epidermal growth factor (HB-EGF) interacts with both EGF-R and a related receptor, ErbB4, whereas transforming growth factor alpha (TGFalpha) interacts only with EGF-R. Because of the unique characteristics of HB-EGF and the potential utility of EGF family members in brain repair, we examine the effects of HB-EGF on rat and mouse CNS cells in vitro and compare them to those of TGFalpha. We find that, like TGFalpha, HB-EGF stimulates the proliferation of CNS astrocytes and multipotent progenitors. These proliferative effects require the expression of EGF-R, as no such effects are observed in cells derived from EGF-R-/- mice. Both HB-EGF and TGFalpha enhanced the survival of neurons derived from the neocortex and the striatum. Within these neuron-enriched cultures, nestin-positive cells but not neurons express EGF-R mRNA, indicating that the neurotrophic actions of EGF-R ligands are a result of indirect stimulation mediated by non-neuronal cells. The neurotrophic actions of HB-EGF and TGFalpha are accompanied by an elevation in immunoreactive dual phosphorylated mitogen-activated protein kinase (MAP kinase) in neurons, providing evidence that the MAP kinase cascade mediates these actions. In situ hybridization studies demonstrate that HB-EGF mRNA is present within the brainstem as early as E14 and subsequently is found in the developing cortical plate, hippocampus, cerebellar Purkinje cells and ventrobasal thalamus, among other brain areas. These findings indicate that HB-EGF may be an important trophic factor in the developing CNS and is a useful candidate molecule for brain repair strategies.

Alterations in BDNF and trkB mRNA levels in the cerebral cortex following experimental brain trauma in rats.

Recent studies have suggested that brain-derived neurotrophic factor (BNDF) and its receptor, trkB, may provide neuroprotection following injury to the central nervous system. Conversely, other studies have implicated BDNF as a contributing factor to neurodegenerative events that occur following injury. In order to further investigate the role of BDNF in neuroprotection, we subjected adult rats to a lateral fluid percussion (FP) injury of moderate severity (2.0-2.1 atm) or sham injury. After survival periods of 1, 3, 6, 24, or 72 h, the brains were processed for the in situ hybridization localization of BDNF and trkB mRNAs using 35S-labeled cRNA probes. Hybridization levels were compared between injured and sham animals for regions of the cortex that were located within, adjacent to, and remote from the site of the cortical contusion. BDNF mRNA levels were significantly decreased in the injured cortex at 72 h, increased in adjacent cortical areas at 3 h, and increased bilaterally in the piriform cortex from 3 to 24 h post-FP injury. Expression of trkB mRNA was significantly decreased at all postinjury time-points in the injured cortex and at 24 h in the adjacent cortex. These results demonstrate that, following lateral FP injury, BDNF and trkB mRNA levels are decreased in cortical regions that contain degenerating neurons, generally unchanged in adjacent regions, and increased in remote areas. Thus, injury-induced decreases in the expression of BDNF and trkB may confer vulnerability to neurons within the cortical contusion.

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study.

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.

Heparin-binding epidermal growth factor-like growth factor in hippocampus: modulation of expression by seizures and anti-excitotoxic action.

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), an EGF receptor ligand, was investigated in rat forebrain under basal conditions and after kainate-induced excitotoxic seizures. In addition, a potential neuroprotective role for HB-EGF was assessed in hippocampal cultures. In situ hybridization analysis of HB-EGF mRNA in developing rat hippocampus revealed its expression in all principle cell layers of hippocampus from birth to postnatal day (P) 7, whereas from P14 through adulthood, expression decreased in the pyramidal cell layer versus the dentate gyrus granule cells. After kainate-induced excitotoxic seizures, levels of HB-EGF mRNA increased markedly in the hippocampus, as well as in several other cortical and limbic forebrain regions. In the hippocampus, HB-EGF mRNA expression increased within 3 hr after kainate treatment, continued to increase until 24 hr, and then decreased; increases occurred in the dentate gyrus granule cells, in the molecular layer of the dentate gyrus, and in and around hippocampal pyramidal CA3 and CA1 neurons. At 48 hr after kainate treatment, HB-EGF mRNA remained elevated in vulnerable brain regions of the hippocampus and amygdaloid complex. Western blot analysis revealed increased levels of HB-EGF protein in the hippocampus after kainate administration, with a peak at 24 hr. Pretreatment of embryonic hippocampal cell cultures with HB-EGF protected neurons against kainate toxicity. The kainate-induced elevation of [Ca2+]i in hippocampal neurons was not altered in cultures pretreated with HB-EGF, suggesting an excitoprotective mechanism different from that of previously characterized excitoprotective growth factors. Taken together, these results suggest that HB-EGF may function as an endogenous neuroprotective agent after seizure-induced neural activity/injury.

Mild experimental brain injury differentially alters the expression of neurotrophin and neurotrophin receptor mRNAs in the hippocampus.

The molecular events responsible for impairments in cognition following mild traumatic brain injury are poorly understood. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), have been identified as having a role in learning and memory. We have previously demonstrated that following experimental brain trauma of moderate severity (2.0-2.1 atm), mRNA levels of BDNF and its high-affinity receptor, trkB, are increased bilaterally in the hippocampus for several hours, whereas NT-3 mRNA expression is decreased. In the present study, we used in situ hybridization to compare BDNF, trkB, NT-3, and trkC mRNA expression in rat hippocampus at 3 or 6 h after a lateral fluid percussion brain injury (FPI) of mild severity (1.0 atm) to sham-injured controls at equivalent time points. Mild FPI induced significant increases in hybridization levels for BDNF and trkB mRNAs, and a decrease in NT-3 mRNA in the hippocampus. However, in contrast to the bilateral effects of moderate experimental brain injury, the present changes with mild injury were restricted to the injured side. These findings demonstrate that even a mild traumatic brain injury differentially alters neurotrophin and neurotrophin receptor levels in the hippocampus. Such alterations may have important implications for neural plasticity and recovery of function in people who sustain a mild head injury.

Differential regulation of neurotrophin and trk receptor mRNAs in catecholaminergic nuclei during chronic opiate treatment and withdrawal.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their receptors trkB and trkC, respectively, are expressed in the locus coeruleus (LC) and ventral tegmental area (VTA), brain regions known to be involved in opiate addiction. Previously, administration of exogenous neurotrophins has been shown to oppose effects of chronic morphine treatment on LC and VTA neurons. However, the response of endogenous neurotrophins in LC and VTA to opiate treatment is unknown. In this study, BDNF, NT-3, trkB, and trkC mRNAs were analyzed in these regions after chronic morphine treatment and during antagonist precipitated withdrawal. Although chronic morphine exposure resulted in only modest increases in BDNF and NT-3 mRNA expression in LC, precipitated withdrawal led to a marked, rapid, and prolonged increase in BDNF mRNA and a delayed decrease in NT-3 mRNA. Levels of trkB and trkC mRNAs, which were unchanged by chronic morphine treatment, were elevated in LC at 2 and 6 hr of withdrawal. By 20 hr, trkB mRNA levels in LC had returned to control, whereas trkC mRNA levels fell below control values. In contrast to the substantial alterations observed in LC, there was no regulation of the neurotrophins or trk mRNAs within the VTA during chronic opiate treatment or withdrawal, with the exception of an increase in trkB mRNA at 6 hr of withdrawal. These results suggest that neurotrophins and their receptors per se may be involved in opiate-induced plasticity of the LC, whereas other mechanisms would appear to be involved in the VTA.

Expression of trkB mRNA is altered in rat hippocampus after experimental brain trauma.

Recent investigations have shown that expression of mRNAs for the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) is differentially altered in the hippocampus following traumatic brain injury. In the present study, modulation of neurotrophin receptor expression was examined in the hippocampus in a rat model of traumatic brain injury using in situ hybridization. Messenger RNA for trkB, the high-affinity receptor for BDNF and neurotrophin-4 (NT-4), was increased between 3 and 6 h bilaterally in the dentate gyrus following a lateral fluid-percussion brain injury of moderate severity (2.0-2.1 atm). No time-dependent alterations were observed for trkB mRNA in hippocampal subfields CA1 and CA3. Levels of mRNA for trkC, the high-affinity receptor for NT-3, did not change in any region of the hippocampus. These data demonstrate that lateral fluid-percussion injury modulates expression of trkB mRNA in the hippocampus and support a role for BDNF/trkB signalling mechanisms in secondary events associated with traumatic brain injury.

Alterations in BDNF and NT-3 mRNAs in rat hippocampus after experimental brain trauma.

Previous studies have suggested that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are neuroprotective or neurotrophic for certain subpopulations of hippocampal neurons following various brain insults. In the present study, the expression of BDNF and NT-3 mRNAs in rat hippocampus was examined after traumatic brain injury. Following lateral fluid percussion (FP) brain injury of moderate severity (2.0-2.1 atm) or sham injury, the hippocampi from adult rats were processed for the in situ hybridization localization of BDNF and NT-3 mRNAs using 35S-labeled cRNA probes at post-injury survival times of 1, 3, 6, 24 and 72 h. Unilateral FP injury markedly increased hybridization for BDNF mRNA in the dentate gyrus bilaterally which peaked at 3 h and remained above control levels for up to 72 h post-injury. A moderate increase in BDNF mRNA expression was also observed bilaterally in the CA3 region of the hippocampus at 1, 3, and 6 h after FP injury, but expression declined to control levels by 24 h. Conversely, NT-3 mRNA was significantly decreased in the dentate gyrus following FP injury at the 6 and 24 h survival times. These results demonstrate that FP brain injury differentially modulates expression of BDNF and NT-3 mRNAs in the hippocampus, and suggest that neurotrophin plasticity is a functional response of hippocampal neurons to brain trauma.

Prenatal ontogeny of the epidermal growth factor receptor and its ligand, transforming growth factor alpha, in the rat brain.

Transforming growth factor alpha (TGF alpha) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGF alpha induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGF alpha and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGF alpha and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGF alpha in many brain structures preceded that of EGF-R mRNA. TGF alpha mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGF alpha mRNA. Northern blot analysis demonstrated that mRNA species for both TGF alpha and EGF-R were similar in embryos and adults. These data indicate that TGF alpha and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain.